A highly sensitive and relatively simple combined gas chromatographic mass spectrometric method was developed to quantify N-arachidonoyl-phosphatidylethanolamine, an anandamide precursor. This method was then successfully applied to measure N-arachidonoylphosphatidylethanolamine in various discrete regions of the rat brain. The N-arachidonoyl-phosphatidylethanolamine was found to be widely and unevenly distributed in the rat CNS. The highest levels were found in spinal cord and striatum and the lowest in cerebellum. Our objective is to use this method to study the regulation of the biosynthesis of N-arachidonoylphosphatidylethanolamine and to assess its physiological role as a precursor of anandamide in specific discrete brain regions.